Fig. 1.
Schematic diagram of the recombination vector for insertion of genes into a vaccinia virus genome using TDS. Plasmid pGPTNEB193 contains the Ecogpt selection gene under the control of the vaccinia virus early/late P7.5 k promoter, a multiple cloning region for the insertion of the sequence to be incorporated into the vaccinia virus genome and the bla gene (not shown) for ampicillin selection of the plasmid in E. coli. For modification of the IBV genome, a sequence corresponding to the region being modified, plus flanking regions of 500 to 800 nucleotides, for recombination purposes is inserted into the multiple cloning sites using an appropriate restriction endonuclease. The plasmid is purified from E. coli and transfected into Vero cells previously infected with a recombinant vaccinia virus containing a full-length cDNA copy of the IBV genome.